TNF-alpha induced apoptosis contributes to the pathophysiology pf many liver diseases. Mitochondria are crucial for signaling TNFalpha-dependent apoptotic cell death in hepatocytes. Recently, we documented that the mitochondrial permeability transition (MPT) mediates mitochondrial depolarization and release of cytochrome c into the cytosol during TNFalpha-induced apoptosis. The MPT is downstream of TNFalpha receptor-mediated activation of caspase 8 and cleavage of Bid, a Bcl-2 family member, to its pro-apoptotic truncated form that translocates to mitochondria. However, a time gap of 7 to 12 hours exists between TNFalpha exposure and the onset of the MPT. The factors responsible for this signaling delay are not understood. This project seeks to determine what other mitochondrial changes must occur to induce the MPT during TNFalpha-induced apoptotic signaling. We hypothesize that: 1) Multiple signals, including ceramide/GD3 formation and Bid and Bax translocation, link upstream cytosolic signaling pathways to a mitochondrial response. 2) Intramitochondrial changes including increased Ca2+, pH, ROS generation and oxidation of mitochondrial GSH and NAD(P)H are also required to induce the MPT, mitochondrial swelling and release of cytochrome c. 3) prior oxidative stress decreases the threshold for onset of the MPT and sensitizes mitochondria to TNFalpha apoptotic signaling, leading to earlier onset of the MPT and apoptosis. To evaluate these hypotheses, we will first measure MPT-related intramitochondrial changes (e.g., Ca2+, pH, ROS and oxidation of NADH and GSH) during TNFalpha-signaling and determine whether these changes regulate the rate and extent of onset of the MPT and subsequent apoptosis. Secondly, we will evaluate how cytosolic factors (ceramides/GD3, Bid and Bax) acts to promote the MPT during TNFalpha signaling. Finally, we will determine the mechanisms by which pre-exposure to an oxidant stress, tert-butylhydroperoxide (t-BuOOH), sensitizes hepatocytes to TNFalpha-induced apoptosis. The results will enhance our understanding of how mitochondria participate in TNFalpha apoptotic signaling and may lead to new therapeutic interventions for liver diseases.